Technology Research & Development Project 7: Cell Specific Proteomics Abstract One of the primary challenges of quantitative proteomics is the dilution of signal due to sampling. Therefore, in this TR&D, we seek to create and refine three workflows to sample 10,000-100,000 cells of specific types prior to proteoform-resolved analysis via top-down proteomics (TDP). We outline a plan of attack involving three common varieties of clinical samples, namely peripheral blood mononuclear cells (PBMCs) (DBP 5), brain sections (DBP 10, 12, and 13), diseased tissue (DBP 12 and 14) and solid tumors (DBP 9). We propose three main strategies to achieve cell specificity and spatial localization, range from actively deployable fluorescence- activated cell sorting (FACS), to the more venerable method of laser capture microdissection (LCM), to a novel, more direct method, which employs a picosecond infrared laser (PIRL) to ablate intact proteins prior to top-down proteomics. We will subsequently analyze collected cell-specific samples by our quantitative TDP platform in both discovery and targeted modes, with the latter utilizing proteoform-specific assays already developed (like for KRAS proteoforms in DBP 9) or the many more to emerge from TR&D 6 throughout the proposed granting period. A specific example derives from our work with PBMCs in DBP 5 (kidney and liver transplant patients), where a panel of 30 proteoforms has emerged from discovery work. From these candidates, we now need determine which immune cells are most responsible for this signal through cell type-resolved sampling. The resulting proteoform-resolved measurements will provide more precise, context-rich, and therefore valuable data revealing how proteins operate in human disease. Although cell-specific TDP technology does not presently exist, its development will unlock significant bottlenecks in protein-level science by sharply improved information about molecular signatures in a proteoform- and cell-specific fashion. While several DBPs will benefit from the sampling of specific cells and tissue regions prior to TDP by FACS, LCM, and PIRL, these technologies are applicable to the spatial sampling of proteins within a wide variety of biological contexts, which will directly benefit a broad range of studies in basic biological, translational and clinical research. The measurement capabilities will also augment the national infrastructure in proteomics, helping to keep pace with an increasingly competitive international landscape in science and technology.